***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****Hi, Giulia,
Great answer! Cn you send us the link to your Nyquist calculator?
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t 04:51 AM 9/22/2016, Giulia De Luca wrote:
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
Hello Andreas,
As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.
Please do not hesitate to contact us if you have any further questions, we are happy to help you!
Best regards,
Giulia
2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:
- ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
- Just quickly:
- * optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).
- Why median?
- Tobias
- Â
- On 9/22/2016 7:22 AM, [hidden email] wrote:
- ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
- Dear all,
- What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/
NyquistCalculator  gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?
- Â
- best wishes
- Â
- Andreas
- Â
- Â
- -----Original Message-----
- From: Craig Brideau [hidden email]
- To: CONFOCALMICROSCOPY [hidden email]
- Sent: Wed, Sep 21, 2016 5:30 pm
- Subject: Re: Shearing Interferometer with Ti-Sapph
- ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
- The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.
- Â
- And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.
- Â
- Craig
- Â
- On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:
- *****
- To join, leave or search the confocal microscopy listserv, go to:
- http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy
- Post images on http://www.imgur.com and include the link in your posting.
- *****
- Hi Tim,
- A shearing interferometer is not going to work with an ultrafast
- laser. There is a note in the thorlabs catalog that can help you
- figure out what it will work with:
- https://www.thorlabs.com/
newgrouppage9.cfm?objectgroup_ id=2970
- The model you have has a 21.16 mm difference in optical pathlength, so
- you will need a coherence length at least that long. Try your laser
- in CW mode, although even that may not be quite 2 cm depending on how
- stable it is without a mode lock. Usually though testing for
- collimation after a beam expander doesn't require an interferometer
- since the beam is large the divergence is low. Have you tried just
- bouncing it across the room and back with a mirror?
- Mike
- On Wed, Sep 21, 2016 at 12:07 AM, Tim <[hidden email]> wrote:
- > *****
- > To join, leave or search the confocal microscopy listserv, go to:
- > http://lists.umn.edu/cgi-bin/
wa?A0=confocalmicroscopy
- > Post images on http://www.imgur.com and include the link in your posting.
- > *****
- >
- > Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
- >
- > Thanks for any help,
- > Tim
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